Properties of biomass powders resulting from the fine comminution of lignocellulosic feedstocks by three types of ball-mill set-up.

Background: Lignocellulosic biomass has many functionalities that hold huge potential for material, energy or chemistry applications. To support advanced applications, the biomass must be milled into ultrafine powder to increase reactivity. This milling unit operation needs to be fully mastered to deliver high-quality standard end-products. Here we studied the relationship between the characteristics of the starting lignocellulosic plant material and the properties of the resulting ultrafine powder in different ball-mill process routes. Methods: Two lignocellulosic biomasses (pine bark and wheat straw) with contrasted compositional and mechanical properties were milled using three ball-mill set-ups delivering different balances of impact force and attrition force. The resulting powders were analysed for particle characteristics (size, agglomeration extent, shape) and powder flow properties (compressibility, cohesion) using a dynamic powder rheometer. Results: Pine bark is more amenable to a fast particle size reduction than the fibrous wheat straw. The resulting pine bark powders appear less compressible but much more cohesive than the straw powders due to particle shape, density and composition factors. The mill set-up working by attrition as dominant mechanical force (vibratory ball mill) produced a mix of large, elongated particles and higher amounts of fines as it acts mainly by erosion, the resulting powder being more prone to agglomerate due to the abundance of fines. The mill set-up working by impact as dominant mechanical force (rotary ball mill) produced more evenly distributed particle sizes and shapes. The resulting powder is less prone to agglomerate due to a preferential fragmentation mechanism. Conclusions: The attrition-dominant mill yields powders with dispersed particle sizes and shapes and the poorest flow properties, while the impact-dominant mill yields more agglomeration-prone powders. The mill set-up working with impact and attrition as concomitant mechanical forces (stirred ball mill) produces powders with better reactivity and flow properties compared to rotary and vibratory mills.

Enzymatic degradation of maize shoots: monitoring of chemical and physical changes reveals different saccharification behaviors.

BACKGROUND: The recalcitrance of lignocellulosics to enzymatic saccharification has been related to many factors, including the tissue and molecular heterogeneity of the plant particles. The role of tissue heterogeneity generally assessed from plant sections is not easy to study on a large scale. In the present work, dry fractionation of ground maize shoot was performed to obtain particle fractions enriched in a specific tissue. The degradation profiles of the fractions were compared considering physical changes in addition to chemical conversion. RESULTS: Coarse, medium and fine fractions were produced using a dry process followed by an electrostatic separation. The physical and chemical characteristics of the fractions varied, suggesting enrichment in tissue from leaves, pith or rind. The fractions were subjected to enzymatic hydrolysis in a torus reactor designed for real-time monitoring of the number and size of the particles. Saccharification efficiency was monitored by analyzing the sugar release at different times. The lowest and highest saccharification yields were measured in the coarse and fine fractions, respectively, and these yields paralleled the reduction in the size and number of particles. The behavior of the positively- and negatively-charged particles of medium-size fractions was contrasted. Although the amount of sugar release was similar, the changes in particle size and number differed during enzymatic degradation. The reduction in the number of particles proceeded faster than that of particle size, suggesting that degradable particles were degraded to the point of disappearance with no significant erosion or fragmentation. Considering all fractions, the saccharification yield was positively correlated with the amount of water associated with [5-15 nm] pore size range at 67% moisture content while the reduction in the number of particles was inversely correlated with the amount of lignin. CONCLUSION: Real-time monitoring of sugar release and changes in the number and size of the particles clearly evidenced different degradation patterns for fractions of maize shoot that could be related to tissue heterogeneity in the plant. The biorefinery process could benefit from the addition of a sorting stage to optimise the flow of biomass materials and take better advantage of the heterogeneity of the biomass.

Automated assay for screening the enzymatic release of reducing sugars from micronized biomass.

BACKGROUND: To reduce the production cost of bioethanol obtained from fermentation of the sugars provided by degradation of lignocellulosic biomass (i.e., second generation bioethanol), it is necessary to screen for new enzymes endowed with more efficient biomass degrading properties. This demands the set-up of high-throughput screening methods. Several methods have been devised all using microplates in the industrial SBS format. Although this size reduction and standardization has greatly improved the screening process, the published methods comprise one or more manual steps that seriously decrease throughput. Therefore, we worked to devise a screening method devoid of any manual steps. RESULTS: We describe a fully automated assay for measuring the amount of reducing sugars released by biomass-degrading enzymes from wheat-straw and spruce. The method comprises two independent and automated steps. The first step is the making of "substrate plates". It consists of filling 96-well microplates with slurry suspensions of micronized substrate which are then stored frozen until use. The second step is an enzymatic activity assay. After thawing, the substrate plates are supplemented by the robot with cell-wall degrading enzymes where necessary, and the whole process from addition of enzymes to quantification of released sugars is autonomously performed by the robot. We describe how critical parameters (amount of substrate, amount of enzyme, incubation duration and temperature) were selected to fit with our specific use. The ability of this automated small-scale assay to discriminate among different enzymatic activities was validated using a set of commercial enzymes. CONCLUSIONS: Using an automatic microplate sealer solved three main problems generally encountered during the set-up of methods for measuring the sugar-releasing activity of plant cell wall-degrading enzymes: throughput, automation, and evaporation losses. In its present set-up, the robot can autonomously process 120 triplicate wheat-straw samples per day. This throughput can be doubled if the incubation time is reduced from 24 h to 4 h (for initial rates measurements, for instance). This method can potentially be used with any insoluble substrate that is micronizable. A video illustrating the method can be seen at the following URL: http://www.youtube.com/watch?v=NFg6TxjuMWU.

Milling itineraries dataset for a collection of crop and wood by-products and granulometric properties of the resulting powders.

Lignocellulosic biomass represents a readily available reservoir of functional elements that can be an alternative to fossil resources for energy, chemicals and materials production. However, comminution of lignocellulosic biomass into fine particles is required to reveal its functionalities, improve its reactivity and allow practical implementation in the downstream processing steps (carrying, dosage, mixing, formulation, shaping…). The sources of lignocellulosics are diverse, with two main families, being agricultural and forest by-products. Due to plant specificity and natural variability, the itineraries of particle size reduction by dry processing, the behavior upon milling and therefore the characteristics of resulting powders can deeply vary according to various raw biomasses [[1], [2]]. This data article contains milling itineraries and granulometric properties of the resulting powders obtained from a collection of by-products from crops (flax fibers, hemp core, rice husk, wheat straw) and woods (pine wood pellets, pine bark, pine sawdust, Douglas shavings, chestnut tree sawdust) representative of currently used lignocellulosic biomass. Samples provided in the form of large pieces (hemp core, pine bark, Douglas shavings) were successively milled using different mills to progressively reduce the matter into coarse, intermediate and finally fine powders. The other samples, supplied as sufficiently small format, were directly processed in the fine powder mill. The machine characteristics and their operating parameters were recorded. The granulometric properties of the powders were analyzed with a laser granulometer and the main indicators related to the particle size distribution (PSD) are presented: (i) d10, d50 (or median diameter) and d90 which are the 10th, 50th and 90th percentiles of the cumulative volume distribution; (ii) the span, which evaluates the width of the particle size distribution; (iii) the calculated specific surface area of the powders which represents the sum of total surface exhibited by the particles per unit of gram and for some powders. The whole particle size distribution of a subset of produced powder samples are also provided for different milling times to illustrate the kinetics of particle size reduction. These data are stored in INRAE public repository and have been structured using BIOREFINERY ontology [3]. These data are also replicated in atWeb data warehouse providing additional query tools [[3], [4]].

Fine Comminution of Pine Bark: How Does Mechanical Loading Influence Particles Properties and Milling Efficiency?

The use of lignocellulosic plant biomass as an alternative to fossil feedstocks for chemistry, energy and materials often involves an intense dry comminution step, for which the energy consumed can vary significantly according to the process parameters, the particle size targeted, and the properties of the biomass. Here we studied the fine milling of maritime pine bark in an impact-mill configuration and in an attrition-mill configuration. The properties of the resulting powders (particle size distribution, particle shape, specific surface area, agglomeration level) obtained in each configuration were compared in relation to process energy consumption. Results evidenced that the agglomeration phenomena drive milling efficiency and limit the possibilities for reaching ultrafine particles. Interestingly, impact loading proved more effective at breaking down coarse particles but tended to generate high agglomeration levels, whereas attrition milling led to less agglomeration and thus to finer particles.

Comminution of Dry Lignocellulosic Biomass: Part II. Technologies, Improvement of Milling Performances, and Security Issues.

Lignocellulosic feedstocks present a growing interest in many industrial processes as they are an ecological alternative to petroleum-based products. Generally, the size of plant raw materials needs to be reduced by milling step(s), to increase density, facilitate transport and storage, and to increase reactivity. However, this unit operation can prove to be important in term of investments, functioning costs, and energy consumption if the process is not fully adapted to the histological structure of the plant material, possibly challenging the profitability of the whole chain of the biomass conversion. In this paper, the different technologies that can be used for the milling of lignocellulosic biomass were reviewed and different avenues are suggested to improve the milling performances thanks to thermal pretreatments. Based on examples on wheat straw milling, the main points to take into consideration in the choice of a milling technologies have been highlighted in regards to the specifications of ground powder. A specific focus on the hazards associated to the milling and the manipulation of fine biomass particles is also realized at the end of the paper from the perspective of industrial applications.

Comminution of Dry Lignocellulosic Biomass, a Review: Part I. From Fundamental Mechanisms to Milling Behaviour.

The comminution of lignocellulosic biomass is a key operation for many applications as bio-based materials, bio-energy or green chemistry. The grinder used can have a significant impact on the properties of the ground powders, of those of the end-products and on the energy consumption. Since several years, the milling of lignocellulosic biomass has been the subject of numerous studies most often focused on specific materials and/or applications but there is still a lack of generic knowledge about the relation between the histological structure of the raw materials, the milling technologies and the physical and chemical properties of the powders. This review aims to point out the main process parameters and plant raw material properties that influence the milling operation and their consequences on the properties of ground powders and on the energy consumption during the comminution.

Cooking behavior of rice in relation to kernel physicochemical and structural properties.

A set of 27 rice varieties were evaluated for their morphological grain characteristics (length, width, thickness, thousand kernel weight, TKW), chemical composition (amylose, protein, and ash content) and starch properties (gelatinization temperature and enthalpy, amylose-lipid complex). In addition, cell walls were characterized by the arabinoxylan and beta-glucan contents. A rapid method for determining optimum rice cooking time was developed based on the swelling ratio; a fixed value of 2.55 gave a gelatinization level of 95% assessed by differential scanning calorimetry and translucence testing. Optimum cooking time appears positively correlated with kernel thickness and TKW but also with ash content. Confocal laser and scanning electron microscope observation of uncooked rice grains revealed different structural features (cell size) and fracture behavior: for some cultivars, the fracture showed ruptured cells, whereas for others most cells were intact. These structural differences, which may be linked to pectin content, could partly explain rice kernel cooking behavior.

Structural heterogeneity of wheat arabinoxylans revealed by Raman spectroscopy.

A set of arabinoxylan samples differing in their arabinose composition and various samples of arabino-xylo-oligosaccharide samples were analysed by Raman spectroscopy. Specific signatures for arabinose substitution were found in several spectral regions, that is, 400-600, 800-950 and 1030-1100 cm(-1). A linear relationship was observed between the peak ratio 855/895 cm(-1) of the second derivative spectra and the A/X ratio determined by chemical analysis. Moreover, spectral changes were observed in the 400-600 cm(-1) region assigned to the coupled vibrations mode in the skeleton: while the intensity of the band at 570 cm(-1) increased with the degree of substitution, that at 494 cm(-1) decreased. Similarly, a linear relationship was observed between the peak intensity ratio 570/494 cm(-1) calculated on the second derivative spectra and the composition data. Analysis of Raman spectra of arabino-xylo-oligosaccharides allowed to identify specific spectral features of disubstitution.

A dry platform for separation of proteins from biomass-containing polysaccharides, lignin, and polyphenols.

License to mill: Proteins were continuously extracted from polysaccharides, lignin, and polyphenol by combining ultrafine milling with electrostatic separation. Such a fractionation process does not involve any solvent, catalyst, or external source of heating. In addition, this dry process is compatible with downstream enzymatic reactions, thus opening an attractive route for producing valuable chemicals from biomass.

A dehydrotrimer of ferulic acid from maize bran.

A new phenolic acid trimer was detected by coupled liquid chromatography/mass spectroscopy in alkali extracts of maize bran. The trimer was purified by preparative silica gel chromatography. The structure of the new compound was elucidated on the basis of 1D and 2D NMR and corresponded to a 4-O-8', 5'-5" dehydrotriferulic acid.

Enzymatic solubilization of arabinoxylans from native, extruded, and high-shear-treated rye bran by different endo-xylanases and other hydrolyzing enzymes.

The overall objective of this research was to find a new way to valorize rye bran, by producing a gellifier from the enzymatic solubilization of arabinoxylans (AX). The effects of three pure endo-xylanases from Aspergillus niger (Xyl-1), Talaromyces emersonii (Xyl-2), and Bacillus subtilis (Xyl-3) and of Grindamyl S100 (GS100), a commercial enzyme preparation containing a Xyl-1 type endo-xylanase, were tested on rye bran to study the solubilization of water-unextractable arabinoxylans (WUAX). Eight different extrusion-treated rye brans were also used as substrates to find the best physical treatment to facilitate enzymatic arabinoxylan (AX) solubilization. Arabinoxylans were better solubilized from the bran extruded at high temperature using Xyl-3. This enzyme was then tested in combination with pure (1,4)-beta-d-arabinoxylan arabinofuranohydrolase (AXH) and endo-beta-d-glucanase or ferulic acid esterase (FAE), from A. niger. Only beta-glucanase in combination with Xyl-3 improved the AX extraction, but it did not have a marked effect on the viscosity of the extracts. Xyl-3 was then tested on a high-shear-treated rye bran, and results were compared to those obtained with the high-temperature-extruded rye bran. The high-shear treatment did not improve the bran AX enzymatic solubilization. The combination of FAE with Xyl-1 or Xyl-3 did not improve the AX extraction from untreated and high-shear-treated rye bran. Finally, to study the gelation capacity of the enzymatically solubilized AX, the effect of the hydrogen peroxide/horseradish peroxidase (H(2)O(2)/POD) was tested on the Xyl-3 high-temperature-extruded bran extracts. Solubilized AX did not gel in the presence of the oxidizing system.

Individual contribution of grain outer layers and their cell wall structure to the mechanical properties of wheat bran.

The mechanical properties of wheat bran and the contribution of each constitutive tissue on overall bran properties were determined on a hard wheat (cv. Baroudeur) and a soft wheat (cv. Scipion). Manual dissection allowed three different layers to be separated from wheat bran, according to radial and longitudinal grain orientations, which were identified by confocal laser scanning microscopy as outer pericarp, an intermediate strip (comprising inner pericarp, testa, and nucellar tissue), and aleurone layer, respectively. Tissue microstructure and cell wall composition were determined. Submitted to traction tests, whole bran, intermediate, and aleurone layers demonstrated elastoplastic behavior, whereas pericarp exhibited elastic behavior. By longitudinal orientation, pericarp governed 50% bran elasticity (elastic strength and rigidity), whereas, in the opposite orientation, bran elastic properties were mostly influenced by the other tissues. Regardless of test orientation, the linear force required to bran rupture corresponded to the sum of intermediate and aleurone layer strengths. According to radial orientation, the intermediate strip governed bran extensibility, but according to longitudinal orientation, all tissues contributed until bran disruption. Tissues from both wheat cultivars behaved similarly. A structural model of wheat bran layers illustrated the detachment of pericarp from intermediate layer within radial bran strips.

New dry technology of environmentally friendly biomass refinery: glucose yield and energy efficiency.

BACKGROUND: Today, most of pretreatments used to convert biomass into biofuels are based on expensive chemical processes that not only do not keep the major components intact after separation, but also consume water and generate many effluents. However, dry fractionation technologies are an important step for future biomass biorefineries since they do not require chemicals and do not generate wastewater. Therefore, the aim of the present study was to evaluate the feasibility of using milling combined with an electrostatic fractionation (ES) of wheat straw (WS) as a way to separate fractions that are enriched in cellulose and more enzymatically accessible, from recalcitrant tissues enriched in lignin-hemicelluloses, in order to produce biofuels. RESULTS: After milling, WS particles are introduced into a tribo-electrostatic separator, where they are positively or negatively charged by tribo-electricity. Then they are introduced into a separation cell comprising two electrodes (+ and -). The negative electrode attracts the positively charged particles and the positive electrode attracts the negatively charged particles. Results show that amorphous cellulose rich particles were clearly more abundant in positively charged fractions (F+), and loose crystalline cellulose, lignin-xylan and ash-containing material were more abundant in negatively charged fractions (F-). Indeed, positively charged fractions (F+) are more accessible upon enzymatic hydrolysis, which resulted, for example, in sugars yield of 43.5% glucose (254 gKg(-1)) for F2B + compared to 25.2% (103 gKg(-1)) for F2A-, and 26.3% (130 gKg(-1)) for unfractionated WS F0, respectively. CONCLUSIONS: The combination strategy of milling and ES fractionation could improve the economic feasibility by low energy consumption (10.5 WhKg(-1)) and it produces reactive lignocelluloses particles with different physicochemical structures, which can be converted easily into biofuels and biomaterials without generating toxic effluents.

Dry fractionation process as an important step in current and future lignocellulose biorefineries: a review.

The use of lignocellulosic biomass is promising for biofuels and materials and new technologies for the conversion need to be developed. However, the inherent properties of native lignocellulosic materials make them resistant to enzymatic and chemical degradation. Lignocellulosic biomass requires being pretreated to change the physical and chemical properties of lignocellulosic matrix in order to increase cell wall polymers accessibility and bioavailability. Mechanical size reduction may be chemical free intensive operation thanks to decreasing particles size and cellulose crystallinity, and increasing accessible surface area. Changes in these parameters improve the digestibility and the bioconversion of lignocellulosic biomass. However, mechanical size reduction requires cost-effective approaches from an energy input point of view. Therefore, the energy consumption in relation to physicochemical properties of lignocellulosic biomass was discussed. Even more, chemical treatments combined with physicochemical size reduction approaches are proposed to reduce energy consumption in this review.

Formation of phenolic microbial metabolites and short-chain fatty acids from rye, wheat, and oat bran and their fractions in the metabolical in vitro colon model.

Rye bran and aleurone, wheat bran and aleurone, and oat bran and cell wall concentrate were compared in their in vitro gut fermentation patterns of individual phenolic acids and short-chain fatty acids, preceded by enzymatic in vitro digestion mimicking small intestinal events. The formation of phenolic metabolites was the most pronounced from the wheat aleurone fraction. Phenylpropionic acids, presumably derived from ferulic acid (FA), were the major phenyl metabolites formed from all bran preparations. The processed rye, wheat, and oat bran fractions contained more water-extractable dietary fiber (DF) and had smaller particle sizes and were thus more easily fermentable than the corresponding brans. Rye aleurone and bran had the highest fermentation rate and extent probably due to high fructan and water-extractable arabinoxylan content. Oat samples also had a high content of water-extractable DF, ß-glucan, but their fermentation rate was lower. Enzymatic digestion prior to in vitro colon fermentation changed the structure of oat cell walls as visualized by microscopy and increased the particle size, which is suggested to have retarded the fermentability of oat samples. Wheat bran was the most slowly fermentable among the studied samples, presumably due to the high proportion of water-unextractable DF. The in vitro digestion reduced the fructan content of wheat samples, thus also decreasing their fermentability. Among the studied short-chain fatty acids, acetate dominated the profiles. The highest and lowest production of propionate was from the oat and wheat samples, respectively. Interestingly, wheat aleurone generated similar amounts of butyrate as the rye fractions even without rapid gas production.

Effects of grinding processes on enzymatic degradation of wheat straw.

The effectiveness of wheat straw fine to ultra-fine grindings at pilot scale was studied. The produced powders were characterised by their particle-size distribution (laser diffraction), crystallinity (WAXS) and enzymatic degradability (Trichoderma reesei enzymatic cocktail). A large range of wheat-straw powders was produced: from coarse (median particle size ∼800 µm) to fine particles (∼50 µm) using sieve-based grindings, then ultra-fine particles ∼20 µm by jet milling and ∼10 µm by ball milling. The wheat straw degradability was enhanced by the decrease of particle size until a limit: ∼100 µm, up to 36% total carbohydrate and 40% glucose hydrolysis yields. Ball milling samples overcame this limit up to 46% total carbohydrate and 72% glucose yields as a consequence of cellulose crystallinity reduction (from 22% to 13%). Ball milling appeared to be an effective pretreatment with similar glucose yield and superior carbohydrate yield compared to steam explosion pretreatment.

Effects of laccase and xylanase on the chemical and rheological properties of oat and wheat doughs.

The effects of Trametes hirsuta laccase and Pentopan Mono BG xylanase and their combination on oat, wheat, and mixed oat-wheat doughs and the corresponding breads were investigated. Laccase treatment decreased the content of water-extractable arabinoxylan (WEAX) in oat dough due to oxidative cross-linking of feruloylated arabinoxylans. Laccase treatment also increased the proportion of water-soluble polysaccharides (WSNSP) apparently due to the beta-glucanase side activity present in the laccase preparation. As a result of the laccase treatment, the firmness of fresh oat bread was increased. Xylanase treatment doubled the content of WEAX in oat dough and slightly increased the amount of WSNSP. Increased stiffness of the dough and firmness of the fresh bread were detected, probably because of the increased WEAX content, which decreased the amount of water available for beta-glucan. The combination of laccase and xylanase produced slight hydrolysis of beta-glucan by the beta-glucanase side activity of laccase and enhanced the availability of AX for xylanase with concomitant reduction of the amount and molar mass of WSNSP. Subsequently, the volume of oat bread was increased. Laccase treatment tightened wheat dough, probably due to cross-linking of WEAX to higher molecular weight. In oat-wheat dough, laccase slightly increased the proportion of WSNSP between medium to low molecular weight and increased the specific volume of the bread. Xylanase increased the contents of WEAX and WSNSP between medium to low molecular weight in oat-wheat dough, which increased the softness of the dough, as well as the specific volume and softness of the bread. The results thus indicate that a combination of laccase and xylanase was beneficial for the textures of both oat and oat-wheat breads.

Arabinoxylan gels: impact of the feruloylation degree on their structure and properties.

Arabinoxylan (AX) samples of decreasing ferulic acid (FA) contents were chemically prepared from water-extractable wheat arabinoxylans without affecting their other structural properties. Gels were obtained from these partially feruloylated WEAX (PF-WEAX) by enzymatic covalent cross-linking of FA leading to the formation of diferulic (di-FA) and tri-ferulic acid (tri-FA). WEAX gelling ability was found related to the WEAX FA content whereas the gel structure and properties depended on the density of newly formed covalent cross-links. FA content of WEAX ranging from 1.4 to 2.3 microg/mg AX gave gels with di-FA cross-links contents from 0.20 to 0.43 microg/mg AX and G' values from 5 to 44 Pa. For WEAX gels with initial FA contents from 1.6 to 2.3 microg/mg AX, average mesh size ranging from 331 to 263 nm were calculated from swelling experiments. Cross-linking densities of gels, determined from swelling experiments, were higher than those that could be theoretically estimated from the di-FA and tri-FA content of WEAX gels. This result suggests that, in addition to di-FA and tri-FA, higher ferulate cross-linking and physical entanglements would contribute to the final WEAX gel structure.